Foliicolous Mycosphaerella spp. and their anamorphs on Corymbia and Eucalyptus

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1 Foliicolous Mycosphaerella spp. and their anamorphs on Corymbia and Eucalyptus Pedro W. Crous 1*, Brett A. Summerell 2, Angus J. Carnegie 3, Caroline Mohammed 4, Winanda Himaman 5 and Johannes Z. Groenewald 1 1 Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands 2 Royal Botanic Gardens and Domain Trust, Mrs. Macquaries Road, Sydney, NSW 2000, Australia 3 Forest Resources Research, NSW Department of Primary Industries, PO Box, Beecroft, NSW 2119, Australia 4 CSIRO Forestry and Forest Products, GPO Box , Hobart 7001, Tasmania 5 Forest Entomology and Microbiology Group, National Park, Wildlife and Plant Conservation Department, Chatuchak, Bangkok, 10900, Thailand Crous, P.W., Summerell, B.A., Carnegie A., Mohammed, C., Himaman, W. and Groenewald, J.Z. (2007). Foliicolous Mycosphaerella spp. and their anamorphs on Corymbia and Eucalyptus. Fungal Diversity 26: The genus Eucalyptus is host to numerous species of Mycosphaerella, several of which are only known as anamorphs, and for which no Mycosphaerella state is known. In this study new Mycosphaerella teleomorph states are described for Nothostrasseria dendritica and Trimmatostroma excentrica. Two new hyphomycete genera are introduced. Of these, Cibiessia gen. nov., with three new species accommodates an arthoconidial synanamorph of Readeriella. Phaeothecoidea gen. nov. is described for species with brown, thick-walled endoconidia. Four additional new species of Mycosphaerella are introduced with several new anamorph species described in Dissoconium, Phaeophleospora, Pseudocercospora, Ramularia and Stenella. Furthermore, an epitype is designated for Mycosphaerella molleriana. This study also presents new Eucalyptus host and distribution records including M. mexicana from Hawaii, M. ohnowa from Australia, M. acaciigena from Australia and Venezuela, M. heimii from Venezuela and Thailand, M. konae from Venezuela, and M. thailandica from Thailand. Key words: Cibiessia, Dissoconium, DNA sequence comparisons, Mycosphaerella, Phaeotheca, Phaeophleospora, Pseudocercospora, Ramularia, Septoria, Stenella, systematics. Introduction The genus Mycosphaerella includes more than 3000 species names (Aptroot, 2006), and several thousand anamorphs that lack known teleomorph * Corresponding author: Pedro Crous; crous@cbs.knaw.nl 143

2 connections (Crous and Braun, 2003). The Mycosphaerellaceae (Capnodiales) (Schoch et al., 2006) includes species that are plant pathogens, saprobes, endophytes (saprobic or plant-pathogenic), or those that have mutualistic (in lichen) associations (Crous et al., 2000, 2001; Verkley et al., 2004). More than species of Mycosphaerella and associated anamorphs have been described from Corymbia and Eucalyptus (Cortinas et al., 2006; Crous et al., 2004b, 2006d, g; Hunter et al., 2006a, b; Andjic et al., 2007; Carnegie et al., 2007). The fact that these host genera are so extraordinarily species-rich in Mycosphaerella spp. might not be surprising, as they include more than 700 species (Brooker and Kleinig, 1994), many of which are known to harbour a wide range of diverse fungal species (Adams et al., 2005; Crous et al., 2006f, b, e; de Beer et al., 2006; Gryzenhout et al., 2006). Although the genera Corymbia and Eucalyptus are indigenous to Australia, many species also occur in other parts of the world (chiefly Eucalyptus), where they are planted as exotics to provide fibre for timber and paper pulp industries. Several species of Mycosphaerella have been associated with Mycosphaerella Leaf Disease (MLD) of eucalypts, causing severe leaf spot, defoliation and shoot die-back (Crous, 1998; Crous et al., 2004b, 2006g; Hunter et al., 2006a, b; Burgess et al., 2007). Many species, however, cause minor leaf spots, rarely resulting in severe disease (Crous et al., 2004b, 2006g, Burgess et al., 2007). Although little is known regarding the host-specificity of Mycosphaerella species, the majority are thought to be highly host-specific. Several recent studies have reported species of Mycosphaerella that are known pathogens of other hosts to be associated with leaf spots of Eucalyptus, where they occurred with other Mycosphaerella spp. (Crous et al., 2004c; Burgess et al., 2007). The co-occurrence of Mycosphaerellaceae on a single leaf spot appears to be a common phenomenon on diverse plant hosts (Crous, 1998; Crous and Groenewald, 2005; Crous et al., 2006a), and it might have led to incorrect assumptions regarding host range and pathogenicity. Crous and Groenewald (2005) also drew attention to the fact that in some cases these species could be major pathogens of hosts other than Myrtaceae. The pogo stick hypothesis was proposed to explain this unusual behavioural pattern, where propagules of a presumed host-specific species show some restricted ability to colonize dead tissue of a non-host, possibly to produce propagules to facilitate onwards dispersal. In Mycosphaerella this behavioural pattern has been observed for teleomorph as well as anamorph states (Crous and Groenewald, unpubl.). This study is part of a series of investigations, in which Mycosphaerella spp. occurring on eucalypts are characterised. The primary aim was to use comparisons of DNA sequence data to clarify obscure anamorph-teleomorph 144

3 connections, and also to recognise new species. These are compared with taxa known in culture and from sequence data, contributing to a global database of Mycosphaerella names, cultures and sequences ( Materials and methods Isolates Mycosphaerella leaf spots were excised, soaked in water for approximately 2 hours, after which they were placed in the bottom of Petri dish lids, with the top half of the dish containing 2% malt extract agar (MEA; Oxoid). Ascospore germination patterns were examined after 24 h, and singleascospore and conidial cultures established as described by Crous (1998). Colonies were sub-cultured onto 2% potato-dextrose agar (PDA; Difco) and oatmeal agar (OA; Gams et al., 2007), and incubated at 25 C under continuous near-ultraviolet light to promote sporulation. DNA isolation, amplification and phylogeny Fungal colonies were established on MEA plates, and genomic DNA was isolated following the protocol of Lee and Taylor (1990). The primers V9G (Hoog and Gerrits van den Ende 1998) and ITS4 (White et al. 1990) were used to amplify part (ITS) of the nuclear rdna operon spanning the 3 end of the 18S rdna gene (SSU), the first internal transcribed spacer (ITS1), the 5.8S rdna gene, the second ITS region and the 5 end of the 28S rdna gene (LSU). The PCR conditions, sequence alignment and subsequent phylogenetic analysis followed the methods of Crous et al. (2004c). To ensure optimal alignment and to simplify the presentation of the trees, the sequence alignment was split into two, whilst keeping phylogenetic lineages together. Sequence data were deposited in GenBank (Table 1) and alignments in TreeBASE (accession number SN3229). Taxonomy Fungal structures were mounted in lactic acid and examined under a light microscope. Wherever possible, 30 measurements ( 0 magnification) were made of structures, with the extremes of spore measurements given in parentheses. Colony colours (surface and reverse) were assessed weekly on PDA, MEA or OA at 25 C in the dark, using the colour charts of Rayner (1970). All cultures obtained in this study are maintained in the culture 145

4 collection of the Centraalbureau voor Schimmelcultures (CBS) in Utrecht, the Netherlands (Table 1). Nomenclatural novelties are listed and descriptions have been deposited in MycoBank < Results and Discussion DNA phylogeny Two alignments of DNA sequences were subjected to phylogenetic analyses. The resulting neighbour-joining trees were congruent for the separate alignments when the substitution models were changed from uncorrected p to the Kimura 2-parameter model and to the HKY85 model as implemented in PAUP. The obtained equally most parsimonious trees mainly differed in the order of taxa at the terminal nodes. The first alignment consisted of 70 taxa including the two outgroups and 512 characters (including alignment gaps) were included in the analyses. Of these characters, 249 were parsimonyinformative, 21 were variable and parsimony-uninformative, and 242 were constant. Parsimony analysis with gaps treated as new states yielded 480 equally most parsimonious trees (TL = 897 steps; CI = 0.586; RI = 0.915; RC = 0.536), one of which is shown in Fig. 1. Although the same lineages were found for the neighbour-joining analyses, the order of the lineages at the deeper nodes differed (data not shown). For example, the clade containing M. fimbriata and Colletogloeopsis spp. is swapped with the clade including M. mexicana and the Readeriella spp. when compared to the figure. The second alignment consisted of 57 taxa including the two outgroups and 494 characters (including alignment gaps) were included in the analyses. Of these characters, 185 were parsimonyinformative, 44 were variable and parsimony-uninformative, and 265 were constant. Parsimony analysis with gaps treated as new state yielded 390 equally most parsimonious trees (TL = 637 steps; CI = 0.597; RI = 0.833; RC = 0.497), one of which is shown in Fig. 2. Similar to the results obtained for the first alignment, the same lineages were found but their order differed in the backbone of the tree. All new species were well-supported, except for the Septoria sp. and Dissoconium eucalypti. The phylogenetic placement suggested by the sequences is discussed in the descriptive notes below each of the treated species. Taxonomy Several anamorph and teleomorph specimens collected in the present study were morphologically and phylogenetically distinct from those presently known. These fungi are described as new taxa as follows: 146

5 Table 1. Isolates of Mycosphaerella spp. and its anamorphs included for sequence analysis and morphological comparison. Teleomorph Anamorph / Synanamorph Strain no. 1 Substrate Country Collector GenBank number Mycosphaerella acaciigena CPC = CBS Eucalyptus sp. Australia B. Summerell EF CPC Eucalyptus camaldulensis E. urophylla Venezuela M.J. Wingfield EF Mycosphaerella associata CPC = CBS Eucalyptus dunnii Australia A.J. Carnegie EF CPC Eucalyptus dunnii Australia A.J. Carnegie EF * CPC = CBS Corymbia henryii Australia A.J. Carnegie EF CPC = CBS Corymbia variegata Australia A.J. Carnegie EF CPC Eucalyptus tereticornis Australia B. Summerell EF Mycosphaerella dendritica Nothostrasseria dendritica * CPC = CBS Eucalyptus deanei Australia B.A. Summerell EF CPC = CBS Eucalyptus nitens Australia A.J. Carnegie EF CPC Eucalyptus deanei Australia B.A. Summerell EF CPC = CBS Eucalyptus globulus Australia C. Mohammed EF Mycosphaerella elongata * CPC = CBS Eucalyptus camaldulensis E. urophylla Venezuela M.J. Wingfield EF Mycosphaerella excentrica Trimmatostroma excentricum * CPC = CBS Eucalyptus agglomerata Australia G. Price EF Mycosphaerella fimbriata CPC = CBS Corymbia sp. Australia P.W. Crous EF * CPC = CBS Corymbia sp. Australia P.W. Crous EF Mycosphaerella heimii CPC = CBS Eucalyptus platyphylla Australia P.W. Crous EF CPC = CBS Eucalyptus urophylla Venezuela M.J. Wingfield EF CPC Eucalyptus urophylla Venezuela M.J. Wingfield EF CPC Eucalyptus urophylla Venezuela M.J. Wingfield EF CPC = CBS Eucalyptus camaldulensis Thailand W. Himaman EF Mycosphaerella konae Pseudocercospora sp. CPC = CBS Eucalyptus camaldulensis Thailand W. Himaman EF Mycosphaerella mexicana CPC = CBS Eucalyptus sp. Hawaii W. Gams EF Mycosphaerella molleriana Colletogloeopsis molleriana * CPC = CBS Eucalyptus sp. Portugal P.W. Crous & EF A.J.L. Phillips Mycosphaerella ohnowa CPC = CBS Eucalyptus dunnii Australia A.J. Carnegie EF Mycosphaerella parkiiaffinis * CPC = CBS Eucalyptus urophylla Venezuela M.J. Wingfield EF Mycosphaerella sp. Cibiessia nontingens / Readeriella sp. * CPC = CBS Eucalyptus tereticornis Australia B. Summerell EF

6 Table 1. Isolates of Mycosphaerella spp. and its anamorphs included for sequence analysis and morphological comparison. Teleomorph Anamorph / Synanamorph Strain no. 1 Substrate Country Collector GenBank number Mycosphaerella thailandica Pseudocercospora thailandica CPC = CBS Eucalyptus camaldulensis Thailand W. Himaman EF CPC Eucalyptus camaldulensis Thailand W. Himaman EF Cibiessia dimorphospora / Readeriella sp. * CPC = CBS Eucalyptus nitens Australia C. Mohammed EF Cibiessia minutispora CPC = CBS Leaf litter of Cussonia sp. South Africa P.W. Crous EF * CPC = CBS Corymbia henryii Australia A.J. Carnegie EF Colletogloeopsis sp. CPC Eucalyptus tereticornis Australia P.W. Crous EF Dissoconium australiensis * CPC = CBS Eucalyptus platyphylla Australia P.W. Crous EF Dissoconium eucalypti * CPC = CBS Eucalyptus tereticornis Australia A. Carnegie EF Phaeophleospora stonei * CPC = CBS Eucalyptus sp. Australia P.W. Crous EF Phaeothecoidea eucalypti * CPC = CBS Eucalyptus botryoides Australia B. Summerell EF Pseudocercospora nogalesii CBS Chamaecytisus proliferus New Zealand C.F. Hill EF Pseudocercospora norchiensis * CPC = CBS Eucalyptus sp. Italy W. Gams EF Ramularia eucalypti * CPC = CBS Eucalyptus grandiflora Italy W. Gams EF CPC = CBS Eucalyptus grandiflora Italy W. Gams EF CPC = CBS Eucalyptus tereticornis Australia P.W. Crous EF Septoria sp. CPC Eucalyptus sp. Italy W. Gams EF CPC = CBS Eucalyptus sp. Italy W. Gams EF Stenella eucalypti * CPC = CBS Eucalyptus tereticornis Australia P.W. Crous EF Stigmina eucalypti * CPC = CBS 121 Corymbia variegata Australia G. Price EF CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS. * Denotes ex-type cultures. 148

7 10 changes Cladosporium colocasiae AF Cladosporium sphaerospermum AF Dissoconium aciculare AF Dissoconium eucalypti CBS AY AY Dissoconium aciculare AY AY Mycosphaerella lateralis AF Dissoconium australiensis CPC AY DQ Mycosphaerella communis CPC AY Mycosphaerella mexicana 90 AY Mycosphaerella readeriellophora AY Readeriella readeriellophora DQ DQ DQ Readeriella eucalypti AY DQ Readeriella mirabilis Cibiessia dimorphospora CBS Cibiessia nontingens CPC CPC CPC Cibiessia minutispora 66 Mycosphaerella dendritica CPC Readeriella novaezelandiae AY CPC CBS Mycosphaerella dendritica CPC CPC CPC Mycosphaerella fimbriata CPC Stigmina eucalypti AF Mycosphaerella pluritubularis DQ Colletogloeopsis dimorpha DQ Colletogloeopsis sp. CPC Colletogloeopsis considenianae DQ Colletogloeopsis gauchensis DQ Coniothyrium zuluense DQ AY AY AY Mycosphaerella molleriana AF AF CPC T Mycosphaerella parkiiaffinis CPC Mycosphaerella sp. AY Mycosphaerella sp. AY Mycosphaerella sp. DQ Mycosphaerella sp. DQ Mycosphaerella ohnowa CPC Mycosphaerella ohnowa AY Mycosphaerella ohnowa AF Mycosphaerella sp. DQ Mycosphaerella sp. DQ Mycosphaerella sp. DQ AY DQ Mycosphaerella parva 91 Phaeothecoidea eucalypti CPC Mycosphaerella excentricum CPC Mycosphaerella pseudosuberosa DQ DQ Mycosphaerella suberosa AY CPC CPC CPC Mycosphaerella associata 60 CPC CPC Mycosphaerella jonkershoekensis DQ Fig. 1. One of 480 equally most parsimonious trees obtained from a heuristic search with random taxon additions of the ITS sequence alignment. The scale bar shows ten changes, and bootstrap support values from 0 replicates are shown at the nodes. Thickened lines indicate the strict consensus branches, and ex-type strains are shown in bold print. The tree was rooted to two Cladosporium species. 149

8 10 changes Cladosporium colocasiae AF Cladosporium sphaerospermum AF Stenella eucalypti CPC Mycosphaerella elongata CPC Phloeospora mimosae-pigrae AJ Mycosphaerella punctiformis AY Ramularia carthami DQ Ramularia collo-cygni AF Ramularia miae DQ CPC CPC Ramularia eucalypti CPC Sonderhenia swartii DQ Sonderhenia walkeri DQ CPC AY AY CPC Mycosphaerella thailandica Mycosphaerella crystallina AF Mycosphaerella holualoana AY Mycosphaerella waimeana AY AY CPC Mycosphaerella acaciigena CPC AF CPC CPC CPC Mycosphaerella heimii 56 CPC CPC AF AY CPC AY Mycosphaerella konae Mycosphaerella brassicicola AF Septoria provencialis DQ Cercospora acaciae-mangii AY Cercospora beticola AY Cercospora apii AY Septoria eucalyptorum DQ Septoria protearum AY Septoria linicola AY CPC CPC Septoria sp. Septoria protearum AY Phaeophleospora stonei CPC Mycosphaerella areola DQ Mycovellosiella eucalypti AF Phaeoramularia saururi AF Pseudocercospora vitis DQ Pseudocercospora luzardii AF Pseudocercospora norchiensis CPC Pseudocercospora nogalesii CBS DQ DQ Mycosphaerella gracilis 64 AY DQ Pseudocercospora pseudoeucalyptorum Fig. 2. One of 390 equally most parsimonious trees obtained from a heuristic search with random taxon additions of the ITS sequence alignment. The scale bar shows 10 changes, and bootstrap support values from 0 replicates are shown at the nodes. Thickened lines indicate the strict consensus branches, and type strains are shown in bold print. The tree was rooted to two Cladosporium species. 150

9 Cibiessia Crous, gen. nov. MycoBank: Etymology: Named for the Centraalbureau voor Schimmelcultures ( CBS ), where the fungus was first discovered by students during a mycological training course. Its unique conidiogenesis captured the imagination of several mycologists who gathered to examine it, and hence the suggestion arose that its name should reflect the unique concentration of mycologically interested persons at CBS. Genus hyphomyceticum ad Mycosphaerellaceas pertinens. Hyphae pallide brunneae, leves, 3 5 μm latae, in conidia dilute brunnea, cylindrica, 0 3-septata, utrinque subtruncata disarticulatae, synanamorphe Readeriella. Hyphomycetous, Mycosphaerellaceae. Hyphae pale brown, smooth, 3 5 µm wide, disarticulating to form pale brown, cylindrical, 0 3-septate conidia with subtruncate ends. A Readeriella synanamorph also formed in culture. Type species: Cibiessia dimorphospora Crous & C. Mohammed, sp. nov. Cibiessia dimorphospora Crous & C. Mohammed, sp. nov. (Fig. 3) MycoBank: Synanamorph: Readeriella sp. Etymology: Name refers to the two asexual states (anamorphs) with different conidial types. Arthroconidia dilute brunnea, cylindrica, utrinque subtruncata, µm, 1( 3)- septata, synanamorphe Readeriellae in vitro formata, conidia ellipsoidea vel subcylindrica, dilute brunnea, µm. Arthoconidia occurring on brown lesions associated with a Pseudocercospora sp. Hyphae pale brown, smooth, 3 5 µm wide, disarticulating at septa to form short, pale brown, cylindrical conidia with obtusely rounded to subtruncate ends; aseptate conidia µm, 1( 3)- septate conidia µm; conidia developing further, becoming medium brown, predominantly aseptate, verruculose, ellipsoidal to subglobose or globose, 5 7 µm diam, with dehiscence scars clearly visible on conidial body; inner layer of the dehiscence scar extends past the outer layer. Readeriella synanamorph: Only observed in culture, and absent in young and older colonies, with Cibiessia state dominant. Conidiomata oozing a dark brown conidial mass; conidiomata pycnidial, subglobose, unilocular; wall consisting of 3 4 layers of brown textura angularis. Conidiophores 0 1-septate, subcylindrical to ampulliform, hyaline to pale brown, smooth, µm, mono- or polyphialide with visible periclinal thickening, or phialide proliferating percurrently near apex; frequently intermingled with cylindrical paraphyses that can extend 5 10 µm above the conidiophores. Conidia narrowly ellipsoid to subcylindrical with rounded ends, pale brown, smooth to finely verruculose, µm. 151

10 Fig. 3. Cibiessia dimorphospora (CBS H-19762). A C. Colonies on PDA. D. Slimy conidial mass of Readeriella state (arrows). E, F. Conidiogenous cells of Readeriella state (arrows). G. Conidia of Readeriella state. H K. Conidia of Cibiessia in vitro. Scale bars = 10 µm. Cultural characteristics: Colonies on PDA slow growing, reaching 30 mm diam after 2 months at 25 C. Surface appearing grey-olivaceous to greenolivaceous due to aerial mycelium and profuse sporulation; margins regular, smooth to slightly feathery; reverse greenish black; young colonies producing a red soluble pigment, but this is inconspicuous in older colonies. Specimen examined: Australia, Tasmania, on Eucalyptus nitens leaves, Oct 2005, C. Mohammed, holotype CBS-H 19762, cultures ex-type CPC = CBS , CPC

11 Notes: Although there are several genera available for species with chains of disarticulating conidia (arthroconidia), none are represented in the Mycosphaerellaceae, and none have ever been linked to Readeriella. As the Readeriella synanamorph of C. dimorphospora rarely occurs in culture, and was not observed on the host, a new genus has been proposed to accommodate the novel arthroconidial anamorph. Species of Cibiessia are present with high bootstrap support (%) in the Readeriella clade. Cibiessia minutispora Crous & Carnegie, sp. nov. (Fig. 4) MycoBank Etymology: Name refers to the conidia that are smaller than those of the other species presently known. Cibiessiae dimorphosporae similis, vel conidia µm. Hyphae pale brown, smooth, 2 3 µm wide, disarticulating at septa to form short, pale brown, cylindrical conidia with obtusely rounded to subtruncate ends; aseptate conidia µm, 1( 2)-septate conidia µm; conidia developing further, becoming medium brown, predominantly aseptate, verruculose, ellipsoidal to subglobose or globose, with dehiscence scars clearly visible on conidial body. Readeriella synanamorph not seen. Cultural characteristics: Colonies flat with even margins, spreading with moderate to prominent aerial mycelium, reaching 25 mm diam after 1 month on PDA; colonies on OA iron-grey, becoming olivaceous-grey on surface due to aerial mycelium; having prominent, diffuse red pigment in agar when cultivated on PDA, colony surface and reverse iron-grey. Specimen examined: Australia, New South Wales, South Grafton, Grafton City Council Landfill Plantation, 152º E, 29º, S, on leaves of Corymbia henryii, 16 Feb. 2006, A.J. Carnegie, holotype CBS-H 19839, Isotype DAR 78030, cultures ex-type CPC = CBS , CPC South Africa, Western Cape Province, Betties Bay, Harold Porter Botanical Garden, leaf litter of Cussonia sp., Jan. 2006, P.W. Crous, CPC = CBS (single ascospore isolate). Notes: Cibiessia minutispora is similar to C. dimorphospora in producing a prominent red pigment in agar, but is distinct due to the absence of a Readeriella synanamorph, and in the fact that it has much smaller conidia. This species is known from two collections, and seems to not be host specific. The South African collection arose from an actively discharged ascospore [using the technique as explained in Summerell et al. (2006), with spores shot upwards onto clean plates], while the Australian isolates occurred with several Mycosphaerella spp. on leaves of E. henryii. 153

12 Cibiessia nontingens Crous & Summerell, sp. nov. (Fig. 5) MycoBank Synanamorph: Readeriella sp. Fig. 4. Cibiessia minutispora (CBS H-19839). A, B. Colonies in vitro on PDA. C G. Conidia and conidiogenous cells in vitro. Scale bar = 10 µm. Teleomorph: Mycosphaerella sp. Etymology: Lacking a red pigment in culture, that is present in other species of the genus presently known. Cibiessiae dimorphosporae similis, sed synanamorphe Readeriellae et pigmento rubro diffundente carens. Leaf spots amphigenous, irregular to subcircular; spots variable from small specks (1 mm diam) to larger spots ( 7 mm diam), or coalescing to form larger blotches, medium brown, with a raised border and thin, red-purple margin. Ascomata pseudothecial, amphigenous, but predominantly epiphyllous, black, subepidermal, globose, up to 90 µm wide; apical ostiole 5 10 µm wide; wall consisting of 2 3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to narrowly ellipsoid to subcylindrical, straight to slightly curved, 8-spored, µm. Ascospores tri- to multi-seriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid-ellipsoidal with obtuse ends, widest just above the septum, medianly 1-septate, constricted at the septum, tapering towards both ends, but 154

13 Fig. 5. Cibiessia nontingens (CBS H-19840). A. Leaf lesion. B. Colonies in vivo. C, D. Asci. E. Asci. F I. Conidia in vitro. Scale bars = 10 µm. more prominently towards the lower end, (9 ) ( 3.5) µm; several ascospores showed remnants of a mucus sheath; no single ascospore cultures were obtained to confirm the anamorph link, though the anamorph formed on top of these ascomata, and the synanamorph among these ascomata. Hyphae pale brown, smooth, 3 5 µm wide, disarticulating at septa to form short, pale brown, cylindrical conidia with obtusely rounded to subtruncate ends; aseptate conidia µm, 1( 3)-septate conidia µm; conidia developing further, becoming medium brown, predominantly aseptate, verruculose, ellipsoidal to subglobose or globose, with dehiscence scars clearly visible on conidial body; inner layer of the dehiscence scar extends past the outer layer. Readeriella synanamorph: Not observed in culture. Conidiomata intermingled among ascomata of a Mycosphaerella sp.; oozing a dark brown conidial mass; conidiomata pycnidial, subglobose, unilocular; wall consisting of 3 4 layers of brown textura angularis. Conidiophores 0 1-septate, subcylindrical to ampulliform, hyaline to pale brown, smooth, µm, 155

14 mono- or polyphialidic. Conidia narrowly ellipsoid to subcylindrical with rounded ends, pale brown, smooth, µm. Cultural characteristics: Colonies flat, spreading, with moderate aerial mycelium and even margins, reaching 40 mm diam after 1 month on OA at 25 C, 50 mm diam on PDA. Colonies on OA olivaceous-grey, on PDA irongrey to greenish black, with numerous mucus droplets on colony surface; colonies greenish black in reverse. Specimen examined: Australia, New South Wales, McWilliam Drive, Douglas Park S E, on leaves of Eucalyptus tereticornis. Open woodland (Cumberland Plains Woodland) of E. molucanna and E. tereticornis on shale derived clay, Jul. 2006, B. Summerell, holotype CBS-H 19840, cultures ex-type CPC = CBS , CPC Notes: Characteristic differences between C. nontingens and C. dimorphospora are the absence of the Readeriella synanamorph in culture, as well as the diffuse red pigment, which are prominent features in the latter species. Key to species of Cibiessia 1. Red pigment produced in colonies on PDA Red pigment absent in colonies on PDA, aseptate conidia up to 10 µm long and 5 µm wide......c. nontingens 2. Readeriella state produced in culture; aseptate Cibiessia conidia up to 7 µm long and 2.5 µm wide... C. dimorphospora 2. Readeriella state not produced in culture; aseptate Cibiessia conidia up to 6 µm long and 3 µm wide... C. minutispora Dissoconium australiensis Crous & Summerell, sp. nov. (Fig. 6) MycoBank Etymology: Named for Australia, the country of origin. Dissoconio communi simile, sed conidiis minoribus, (20 )23 25( 27) (3 )4( 5) µm, distinguendum. Mycelium internal and external, consisting of branched, septate, smooth, hyaline to pale brown hyphae, 2 3 µm wide. Conidiophores separate, arising from hyphae, subcylindrical, subulate or lageniform, tapering to a bluntly rounded or truncate apex, straight to curved, smooth, medium brown, aseptate, µm; loci terminal and lateral, indistinct. Conidia (20 )23 25( 27) (3 )4( 5) µm, solitary, pale olivaceous-brown, smooth, ellipsoid to obclavate, 1-septate, apex obtuse, base obconic-truncate, hilum unthickened, µm wide. Secondary conidia not observed on MEA or on SNA. Cultural characteristics: Colonies on MEA reaching 30 mm diam after 1 month at 25 C; erumpent with sparse aerial mycelium, hazel to isabelline, with feathery margins; umber in reverse. 156

15 Specimen examined: Australia, Queensland, Cairns, nr Kuranda, S , E , on leaves of Eucalyptus platyphylla, 26 Aug. 2006, P.W. Crous, holotype CBS- H 19837, culture ex-type CPC = CBS Notes: Morphologically and phylogenetically D. australiensis is similar to D. commune and D. dekkeri (= M. lateralis). Conidia of D. australiensis (20 ) 23 25( 27) (3 )4( 5) µm are on average smaller than those of D. commune ( µm, av µm), and somewhat larger than the common range of D. dekkeri (15 )17 21( 35) (2 )3.5 4( 4.5) µm (Crous, 1998). Although the present strain failed to produce microconidia on various culture media, this cannot be seen as a species character until more strains have been collected and studied. Fig. 6. Dissoconium australiensis (CBS H-19837). Conidia and conidiogenous cells in vitro. Scale bar = 10 µm. Dissoconium eucalypti Crous & Carnegie, sp. nov. (Fig. 7) MycoBank Etymology: Named after its host plant, Eucalyptus. Dissoconia aciculari simile, sed conidiis primariiss minoribus, (8 )10 12( 14) (4.5 ) 5 6 µm, secondariis majoribus, µm, differens. Mycelium internal and external, consisting of branched, septate, smooth, hyaline to pale brown hyphae, 2 3 µm wide. Conidiophores separate, arising from hyphae, subcylindrical, subulate or lageniform, tapering to a bluntly rounded or truncate apex, straight to once geniculate, smooth, medium brown, aseptate, µm; loci terminal and lateral, visible as slightly 157

16 Fig. 7. Dissoconium eucalypti (CBS H-19770). Conidia and conidiogenous cells in vitro. Scale bar = 10 µm. thickened, darkened scars, µm wide. Conidia (8 )10 12( 14) (4.5 )5 6 µm, solitary, pale olivaceous-brown, smooth, ellipsoid to obclavate, 1- septate,apex obtuse, base obconic-truncate, hilum thickened, somewhat darkened, µm wide. Secondary conidia developing adjacent to primary 158

17 conidia, pale olivaceous to subhyaline, aseptate, pyriform, with a truncate base, µm; anastomosing with primary conidia after active discharge. Cultural characteristics: Colonies on MEA reaching 15 mm diam after 3 weeks at 25 C; erumpent with sparse aerial mycelium, buff to olivaceous-buff, with feathery margins; cinnamon in reverse. Specimen examined: Australia, New South Wales, Morpeth Park, Plantation, Bonalbo, 152º E, 28º 46 3, on leaves of Eucalyptus tereticornis, 8 Feb 2006, A. Carnegie, holotype CBS-H 19770, cultures ex-type CPC = CBS , CPC Notes: Although several species of Dissoconium have been described from Eucalyptus (Crous et al., 2004b), D. eucalypti is distinct in having smaller primary and larger secondary conidia than those species known to date. Phylogenetically it clusters close to the ex-type strain of D. aciculare, which has larger primary ( µm), and secondary ( µm) conidia (De Hoog et al., 1983). However, D. eucalypti differs with 5 nucleotides in the ITS1 region when compared to strains identified as D. aciculare. Mycosphaerella associata Crous & Carnegie, sp. nov. (Fig. 8) MycoBank Etymology: Name refers to its co-occurrence with other species of Mycosphaerella on the same leaf spots. Ascosporae fusoideae-ellipsoideae, (12 )13 16( 17) (3.5 )4 5( 6) µm. Leaf spots amphigenous, irregular to subcircular, 4 6 mm diam, medium brown, with a thin, raised, dark brown border on the adaxial surface; dark brown with patches of grey due to the lifting cuticle on the abaxial surface, displaying numerous small cracks within the lesion tissue. Ascomata pseudothecial, amphigenous, but predominantly hypophyllous, black, subepidermal to erumpent, globose, up to 120 µm wide; apical ostiole µm wide; wall consisting of 2 3 layers of medium brown textura angularis. Asci aparaphysate, but with remains of hamathecium visible, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoidal, straight to slightly curved, 8-spored, µm. Ascospores tri- to multi-seriate, overlapping, hyaline, guttulate, thick-walled, straight, fusoid-ellipsoidal with obtuse ends, widest in middle of apical cell, medianly 1-septate, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (12 ) 13 16( 17) (3.5 )4 5( 6) µm; ascospores with persistent mucus sheath. Ascospores germinate from polar ends, with germ tubes parallel to the long axis of the spore; spore distorting and becoming prominently constricted at the septum, verruculose and brown; germ tubes pale brown, not straight and even, but irregularly crenate, 5 8 µm wide, at times developing 1 2 additional spore septa and additional germ tubes (germination Type H sensu Crous, 1998). 159

18 Fig. 8. Mycosphaerella associata (CBS H-19833). A. Leaf spot. B. Asci. C E. Germinating ascospores. F. Ascospores. Scale bars = 10 µm. Cultural characteristics: Colonies erumpent with moderate aerial mycelium; margins catenulate, smooth; surface uneven on OA, olivaceous-grey with patches of pale olivaceous-grey to iron-grey, reaching 15 mm diam after 1 month at 25 C (on OA and PDA); on PDA olivaceous-grey with patches of pale olivaceous-grey to grey-olivaceous. Specimens examined: Australia, New South Wales, South Grafton, Grafton City Council Landfill Plantation, 152º E, 29º, S, on leaves of Corymbia henryii, 16 Feb. 2006, A.J. Carnegie, holotype CBS-H 19833, isotype DAR 78031, cultures ex-type CPC = CBS , CPC (occurring with Lembosina sp.); New South Wales, Bungawalbin, Robertson Plantation, 153º E, 29º 5 34 S, on leaves of Corymbia variegata, 23 Jan. 2005, A.J. Carnegie, DAR 78032, cultures CPC = CBS , CPC (occurring with Lembosina sp.); New South Wales, Bungawalbin, Robertson Plantation, 153º E, 29º 5 34 S, on leaves of Eucalyptus dunnii, 14 Feb. 2006, A.J. Carnegie, cultures CPC = CBS , CPC , (occurring with M. suberosa). Notes: Mycosphaerella associata frequently colonizes lesions of other ascomycetes, but it also occurs singly. Although several species have overlapping ascospore dimensions that overlap with those of M. associata (Crous, 1998), none share its rather unique mode of ascospore germination. 160

19 Mycosphaerella jonkershoekensis (GenBank DQ302968) clusters with % bootstrap support in the M. associata clade, but differs with one nucleotide in both the ITS1 and ITS2 region from this species. Mycosphaerella dendritica Crous & Summerell, sp. nov. (Fig. 9) MycoBank Anamorph: Nothostrasseria dendritica (Hansf.) Nag Raj, Can. J. Bot. 61: (Basionym) Spilomyces dentriticus Hansf., Proc. Linn. Soc. N. S. W. 81: Ascosporae fusoideae-ellipsoideae, (11 )12 13( 15) 3 3.5( 4.5) µm, anamorphe Nothostrasseria dendritica formata in vitro. Leaf spots amphigenous, irregular to subcircular, 2 8 mm diam, pale to gray-brown, with raised borders and thin, dark brown margins. Ascomata pseudothecial, amphigenous, black, subepidermal, globose, up to 150 µm wide; wall consisting of 2 3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, broadly ellipsoid, straight to slightly curved, 8-spored, µm. Ascospores bi- to triseriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid-ellipsoidal with obtuse ends, widest just above the septum, medianly 1-septate, not to slightly constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (11 )12 13( 15) 3 3.5( 4.5) µm; encased in a mucus sheath. Ascospores germinate irregularly, but mostly from polar ends, with germ tubes parallel to the long axis, but also with secondary germ tubes forming on the spore, at right angles to the long axis of the spore (Type D or I, sensu Crous 1998); spore distorting, becoming constricted, but remaining hyaline, µm diam. Conidiomata black, globose, pycnidial, scattered, immersed in leaf tissue, but immersed to almost superficial on agar, up to 250 µm diam. Conidiophores ampulliform to lageniform, hyaline, smooth, 0 1- septate, mono- to polyphialidic, rarely proliferating percurrently, rarely branched, with loci terminal but also lateral, µm. Conidia consisting of an ellipsoid body with obtuse apex, tapering to a tubular basal appendage; body medium brown, verruculose, µm; tubular appendage separated from the conidium body by a septum, unbranched, hyaline, smooth, µm. Cultural characteristics: Colonies on PDA reaching 35 mm diam after 5 weeks at 25 C; colonies erumpent, with moderate, woolly aerial mycelium, pale olivaceous-grey to olivaceous-grey, margins smooth, regular; reverse iron-grey with zones of olivaceous-grey; colonies produce a faint, diffuse, pink pigment in agar. Colonies form numerous erumpent, black, globose, dark brown to black conidiomata on PDA and MEA. 161

20 Fig. 9. Mycosphaerella dendritica and its anamorph, Nothostrasseria dendritica (CBS H ). A. Leaf spot. B, C. Colonies on MEA (arrows indicate conidiomata). D F. Asci. G, H. Ascospores (arrow indicates sheath). I, J. Germinating ascospores. K, L. Conidiogenous cells. M, N. Conidia. Scale bars: E, F, K, N = 10, G J = 3.5 µm. Specimens examined: Australia, New South Wales, Wollemi National Park, on leaves of Eucalyptus deanei, Feb 2006, B.A. Summerell, holotype CBS-H 19772, cultures ex-type CPC = CBS , CPC ; New South Wales, Laurel Hill, Bago State Forest, research trial, on leaves of E. nitens, 22 Dec. 2005, A.J. Carnegie, CPC = CBS ; Tasmania, on leaves of E. globulus, 31 Aug. 2006, C. Mohammed, CPC = CBS , CPC Notes: As far as we could establish, this is the first record of Nothostrasseria dendritica grown in pure culture. This is also the first record of 162

21 its teleomorph, which is a species of Mycosphaerella, described here as M. dendritica. Phylogenetically Nothostrasseria clusters with species of Readeriella, but is different from R. novaezelandiae at three nucleotide positions in the ITS1 region and one in the ITS2 region. Although species of Readeriella have brown conidia that have up to three obtuse, apical projections, they lack basal appendages, and are thus tentatively retained as separate genera. The conidiogenesis of both genera is, however, similar, with conidia forming on mono- or polyphialides, which can also proliferate percurrently. Mycosphaerella elongata Crous & M.J. Wingf., sp. nov. (Fig. 10) MycoBank Etymology: Named after its characteristic long ascospores. Ascosporae fusoideae-ellipsoideae, (18 )20 25 (4 )4.5( 5) µm. Leaf spots amphigenous, irregular to subcircular, 3 13 mm diam, medium brown, with a thin, raised, dark brown to red-brown border. Ascomata pseudothecial, amphigenous, but predominantly epiphyllous, dark brown, subepidermal to somewhat erumpent, globose, up to 150 µm wide; apical ostiole up tp 30 µm wide; wall consisting of 2 3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoidal, straight to slightly curved, 8-spored, µm. Ascospores tri- to multi-seriate, overlapping, hyaline, guttulate, thin-walled, straight to slightly curved, fusoid-ellipsoidal with obtuse ends, widest in middle of apical cell, 1-septate, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (18 )20 25 (4 )4.5( 5) µm; basal cell frequently 1 4 µm longer than apical cell. Ascospores germinate from both ends, with germ tubes parallel to the long axis of the spore; spore not darkening, nor distorting, becoming up to 5 µm wide (germination Type C sensu Crous, 1998). Cultural characteristics: Colonies on MEA erumpent, convex, radially striated; margins smooth, even; surface cinnamon with patches of pale vinaceous aerial mycelium in centre; reverse brown-vinaceous; reaching 11 mm diam after 2 months at 25 C. Specimen examined: Venezuela, El Piñal Lotes farm near Acarigua, on leaves of Eucalyptus camaldulensis urophylla, Oct. 2006, M.J. Wingfield, holotype CBS-H 19824, cultures ex-type CPC = CBS , CPC Notes: Mycosphaerella elongata has characteristically long ascospores (up to 25 µm long), somewhat reminiscent of M. longibasalis ( µm; Crous, 1998), but shorter. It also has different lesions, with those of M. longibasalis being pale brown in colour. 163

22 Fig. 10. Mycosphaerella elongata (CBS H-19824). A. Leaf spot. B D. Asci and ascospores. E G. Germinating ascospores. Scale bar = 10 µm. Mycosphaerella excentrica Crous & Carnegie, sp. nov. (Fig. 11) MycoBank Anamorph: Trimmatostroma excentricum B. Sutton & Ganap., N.Z. J. Bot. 16: Ascosporae fusoideae-ellipsoideae, (10 )15 18( 23) (3 )4 µm, anamorphe Trimmatostroma excentrica formata in vitro. Leaf spots amphigenous, irregular, corky, medium to dark brown, raised, with an irregular margin and thin, red-brown border, 2 12 mm diam. Ascomata amphigenous, separate, dark brown, subepidermal, becoming superficial, globose, up to 160 µm wide; apical ostiole up to 20 µm wide, but frequently opening by means of irregular rupture; wall of 2 3 layers of dark brown, thickwalled textura angularis. Asci fasciculate, bitunicate, aparaphysate (through remains of the hamathecium observed in some ascomata), 8-spored, obovoid to broadly ellipsoidal, straight to slightly incurved, µm. Ascospores 164

23 Fig. 11. Mycosphaerella excentrica and its anamorph Trimmatostroma excentricum (CBS H ). A. Leaf spot. B, C. Asci. D. Ascospores. E. Colony on OA. F, G. Conidia and conidiogenous cells in vitro. Scale bars = 10 µm. tri to multiseriate, fusoid-ellipsoidal with obtuse ends, hyaline, smooth, but pale brown and verruculose in old asci, becoming 3-septate, not constricted at median septum, thick-walled, guttulate, widest in the middle of the apical cell, with persistent mucous sheath, (10 )15 18( 23) (3 )4 µm. Conidia in vitro formed in basipetal chains, smooth, medium brown, 4-celled, consisting of two basal cells with truncate lateral sides (adhesion scars present when catenulate), each giving rise to a secondary globose apical cell, that can extend and develop two additional septa in some cases; primary cells µm, secondary cells µm wide, 4 6 µm long, but with additional septa these arms can become up to 15 µm long (excluding the basal cell); septa separating the primary and secondary cells are dark-brown and thick-walled. Cultural characteristics: Colonies on OA erumpent, black, powdery, uneven with catenulate margins; aerial mycelium absent, reaching 10 mm diam after 2 months on OA at 25 C; fertile forming anamorph. Specimen examined: Australia, New South Wales, Mackenzie Creek Road, Kempsey, Byrne Plantation, 152º E, 30º S, on leaf spots of E. agglomerata, 13 Apr. 2005, 165

24 G. Price, holotype CBS-H 19829, isotype DAR 78033, culture ex-type CPC = CBS Notes: No teleomorph has previously been linked to T. excentricum, and this is the first record of this species grown in pure culture. Although the anamorph has a different conidial morphology to those of other species of Trimmatostroma, it clusters with other members of the genus based on its DNA phylogeny. Mycosphaerella fimbriata Crous & Summerell, sp. nov. (Fig. 12) MycoBank Etymology: Named after its characteristic leaf spots with radiating hyphal strands. Ascosporae obovoidea, (18 )22 17( 30) (6 )7( 8) µm. Leaf spots amphigenous, irregular to circular, 5 15 mm diam, medium to dark brown, with radiating superficial mycelium, spreading from ascomata that are predominantly in the middle of the lesion; hyphae red-brown, 5 8 µm wide, thick-walled, verruculose, aggregating in hyphal strands (also in vitro), with chlamydospore-like cells, up to 15 µm diam, aggregating in clusters; forming spermatogonia in the outer region of the lesion (also formed in vitro). Ascomata pseudothecial, amphigenous, black, subepidermal, but becoming erumpent, globose, up to 120 µm wide; apical ostiole µm wide; wall consisting of 6 8 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to broadly ellipsoidal, straight to slightly curved, 8-spored, with the endotunica having 3 5 well differentiated layers, visible when mounted in clear lactic acid, µm. Ascospores multiseriate, overlapping, hyaline, guttulate, thin-walled, straight to slightly curved, obovoid, with obtuse ends, widest near the apex of the apical cell, mostly medianly 1-septate, constricted at the septum; larger ascospores tend to be unequally 1-septate, with the upper cell being up to 13 µm long, and the bottom cell up to 17 µm long, tapering towards both ends, but more prominently towards the lower end, (18 )22 17( 30) (6 )7( 8) µm; ascospores frequently with a persistent mucous sheath. Ascospores germinate from both ends, but not necessarily polar, with 2 4 germ tubes more or less parallel to the long axis of the spore (or germ tubes 3 4 irregular); original spore becoming transversely septate, constricted, with mucus sheath prominently visible; spore becoming up to 10 µm wide, darkening and becoming verruculose (germination Type I sensu Crous, 1998). Cultural characteristics: Colonies on MEA slow growing, reaching 5 mm diam after 2 months; colonies erumpent, with moderate aerial mycelium and uneven, feathery margins; surface olivaceous-grey, at times fawn in centre due to superficial mycelium; reverse dark-brick. On OA erumpent, spreading with 166

25 Fig. 12. Mycosphaerella fimbriata (CBS H-19828). A, B. Leaf spots with radiating superficial hyphae. C. Hyphal strands. D. Chlamydospore-like structures. E. Colony on OA. F H. Asci, with layered endotunica. I, J, N. Germinating ascospores. K M. Ascospores. Scale bars: F = 7, I = 10 µm. even, smooth margins; surface dark-brick in centre, outer zone olivaceous-grey, forming a diffuse, dark-vinaceous pigment in the agar, reaching 10 mm diam 167

26 after 2 months at 25 C; colonies forming numerous spermatogonia when inoculated onto OA. Specimen examined: Australia, Queensland, Cairns, S , E , on leaves of Corymbia sp., 26 Aug. 2006, P.W. Crous, holotype CBS-H 19828, cultures extype CPC = CBS , CPC ; Cairns, Mareeba Wetlands, Peninsula Development Road, S , E , 380 m, on leaves of Corymbia sp., 27 Aug. 2006, P.W. Crous, CBS-H 19827, cultures CPC = CBS , CPC Notes: Mycosphaerella fimbriata is unique among the species known on Eucalyptus in having distinct brown leaf spots covered by strands of red-brown, radiating hyphae, and having a multi-layered ascal endotunica. Phylogenetically it is related to Stigmina eucalypti and Colletogloeopsis spp. Mycosphaerella parkiiaffinis Crous & M.J. Wingf., sp. nov. (Fig. 13) MycoBank Etymology: Name refers to its morphology which is similar to that of Mycosphaerella parkii. Ascosporae fusoideae-ellipsoideae, (8 )9 10 3( 3.5) µm. Leaf spots amphigenous, irregular to subcircular, 6 30 mm diam, pale to medium brown, with a thin, raised, dark brown border, and a red-purple margin. Ascomata pseudothecial, amphigenous, dark brown, subepidermal to somewhat erumpent, globose, up to 80 µm wide; apical ostiole µm wide; wall consisting of 2 3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate, subsessile, obovoid to ellipsoidal, straight to slightly curved, 8-spored, µm. Ascospores tri- to multi-seriate, overlapping, hyaline, guttulate, thin-walled, straight, fusoid-ellipsoidal with obtuse ends, widest in middle of apical cell, medianly 1-septate, constricted at the septum, tapering towards both ends, but more prominently towards the lower end, (8 )9 10 3( 3.5) µm. Ascospores germinate from both ends, with germ tubes parallel or irregular to the long axis of the spore, with 2 4 germ tubes developing; spore not darkening, but distorting, becoming up to 6 µm wide (germination Type D sensu Crous, 1998). Cultural characteristics: Colonies on MEA reaching 18 mm diam after 2 months at 25 C; colonies erumpent, spreading, with moderate aerial mycelium, and smooth, but somewhat feathery margins; surface olivaceous-grey in the centre, pale olivaceous-grey in outer region; reverse olivaceous-black. Specimen examined: Venezuela, near Acarigua, on leaves of Eucalyptus urophylla, Oct. 2006, M.J. Wingfield, holotype CBS-H 19823, cultures ex-type CPC = CBS , CPC Notes: In comparison to other Mycosphaerella spp., M. parkiiaffinis has small, nondescript spores, and an irregular ascospore germination pattern (Type D), similar to species in the M. parkii complex. It is distinct in lacking a 168

27 Fig. 13. Mycosphaerella parkiiaffinis (CBS H-19823). A. Leaf spot. B. Colony on MEA. C, D. Asci. E. Ascospores. F H. Germinating ascospores. Scale bar = 10 µm. Stenella anamorph, and having smaller ascospores than those of M. parkii,which are up to 15 µm long (Crous, 1998; Crous et al., 2006g). Phylogenetically, it is most closely related to M. ohnowa. Phaeophleospora stonei Crous, sp. nov. (Fig. 14) MycoBank Etymology: Named for Dr. Jeff Stone, who collected this fungus with P.W.C. along the river bank in Kuranda before the IMC8 congress. Phaeophleosporae lilianae similis, sed conidiis minoribus, (25 )30 33( 35) (3.5)4( 5) µm, distinguenda. Leaf spots amphigenous, circular to subcircular, pale brown with a raised, dark brown border, and thin, red-purple margin, 1 4 mm diam. Conidiomata amphigenous, subepidermal with a central ostiole, from where conidia exude in a brown cirrus; scattered, globose, dark brown, up to 200 µm diam; wall of 3 4 layers of dark brown textura angularis. Conidiogenous cells pale brown, 169

28 Fig. 14. Phaeophleospora stonei (CBS H-19835). A, B. Leaf spots (arrow indicates conidial cirrus). C. Colony on OA. D, E. Conidiogenous cells and conidia. F. Conidia. Scale bar = 10 µm. smooth, ampulliform to doliiform, µm, proliferating percurrently near apex. Conidia subcylindrical to narrowly obclavate, widest at basal septum, tapering to a subtruncate, flattened hilum with minute marginal frill, and tapering in the apical cell to an obtuse apex; cellular content granular to not so in vivo, conidia 3( 6)-euseptate (septa appear thicker in Shear s than in clear lactic acid, but never distoseptate); conidia guttulate and darker brown in vitro, but similar in dimensions, (25 )30 33( 35) (3.5)4( 5) µm. Cultural characteristics: Colonies slow-growing, reaching 7 mm diam on OA after 2 months at 25 C; erumpent, with moderate aerial mycelium and uneven, but smooth margins, pale mouse-grey to olivaceous-grey. Specimen examined: Australia, Queensland, Cairns, Kuranda, Karoomba River Walk, S , E , on leaves of Eucalyptus sp., 19 Aug. 2006, P.W. Crous & J. Stone, holotype CBS-H 19835, culture ex-type CPC = CBS , CPC Notes: Swart and Walker (1988) erected the genus Sonderhenia to separate taxa with distoseptate conidia from those with transversely euseptate 170

29 conidia. Walker et al. (1992) placed several similar taxa with eu-septate conidia in a new genus, Kirramyces. Crous et al. (1997) treated Kirramyces as synonym of Phaeophleospora. The type species of Kirramyces (K. epicoccoides) clusters apart from that of Phaeophleospora (P. eugeniae) within the Mycosphaerellaceae. The fact that P. stonei does not cluster with the type of Phaeophleospora nor Kirramyces, suggests that Phaeophleospora is polyphyletic, as are most anamorph genera in the Mycosphaerellaceae. The phylogenetic analysis places it closest to M. areola and Pseudocercospora spp. Phaeothecoidea Crous, gen. nov. MycoBank Etymology: Its characteristic endoconidia resemble Phaeotheca, but the structures are always dark and thick-walled. Phaeothecae similis, sed structuris omnino fuscis et crassitunicatis. Hyphomycetous, Mycosphaerellaceae. Hyphae pale to medium brown, verruculose, 4 6 µm wide, end cells dividing into several endoconidia. Endoconidia pale to medium brown, verruculose, thick-walled, ellipsoid to obovoid, obclavate or irregularly triagular, µm, becoming 1( 2) septate, medium to dark brown, verruculose to verrucose, µm, giving rise to additional endoconidia. Type species: Phaeothecoidea eucalypti Crous & Summerell., sp. nov. Phaeothecoidea eucalypti Crous & Summerell, sp. nov. (Fig. 15) MycoBank Etymology: Named after its host genus, Eucalyptus. Conidia matura brunnea, verruculosa, crassitunicata, ellipsoidea vel irregulariter triangualria, μm. Hyphae in vitro creeping, subhyaline, verruculose, branched, septate, 4 6 µm wide, becoming swollen, up to 15 µm wide, verruculose, medium brown; end cells dividing into several endoconidia, which are released upon rupture of the cell wall. Endoconidia pale to medium brown, verruculose, thick-walled, ellipsoid to obovoid, obclavate or irregularly triagular, µm after liberation; swelling, becoming 1( 2) septate, medium to dark brown, verruculose to verrucose, µm; conidia giving rise to 1 2( 4) additional endoconidia, with outer wall of primary conidium visible as prominent collarette around endoconidia during rupture, and on outer wall of primary conidium after conidial release. Cultural characteristics: Colonies on OA and PDA black, slimy, shiny, irregular, elevated with a catenulate margin, lacking aerial mycelium, but having slimy droplets on the surface; growing 5 mm diam in 3 weeks on OA, 1 cm on PDA. 171

30 Fig. 15. Phaeothecoidea eucalypti (CBS H-19836). A. Colonies on OA. B, C. Hyphal ends with endoconidia. D F. Conidia. Scale bar = 10 µm. Specimen examined: Australia, New South Wales, Clareville Beach Reserve, on leaves of Eucalyptus botryoides, Feb. 2006, B. Summerell, holotype CBS-H 19836, culture ex-type CPC = CBS Notes: The genus Melanothecoidea is reminiscent of the genera Hyphospora (teleomorph: Cumminutispora) and Phaeotheca, which both have endoconidia, and are placed in the Dothideomycetes. However, neither of these genera cluster within Mycosphaerella, and they are also morphologically distinct by tending to have more thin-walled conidia, that become pigmented with age (Zalar et al., 1998). Melanothecoidea eucalypti clusters among species of Trimmatostroma within Mycosphaerella, but is distinct in that members of Trimmatostroma generally have dry, disarticulating conidia, while colonies of Melanothecoidea are wet and slimy, and have endoconidia. The phylogenetic analysis places it closest to M. excentrica and M. suberosa. Pseudocercospora norchiensis Crous, sp. nov. (Fig. 16) MycoBank Etymology: Named after the type locality in Italy, Norchia, Prov. Viterbo. Conidia obclavata, (0 )5 9( 12)-septata, (50 )80 120( 140) (5 )6( 7) µm. 172

31 Fig. 16. Pseudocercospora norchiensis (CBS H-19841). A. Conidiophores. B F. Conidia. Scale bar = 10 µm. Leaf spots amphigenous, irregular to subcircular, 2 6 mm diam, medium brown with a raised border, and a thin red-purple margin. Mycelium internal, smooth, consisting of branched, septate, smooth, pale brown hyphae, 3 4 µm wide; superficial mycelium developing once incubated in moist chambers. Caespituli fasciculate, epiphyllous, pale brown on leaves, up to 160 µm wide and 150 µm high. Conidiophores aggregated in highly dense fascicles arising from the upper cells of a brown stroma up to 160 µm wide and 90 µm high; conidiophores medium brown, smooth, 3 5-septate, subcylindrical, straight to variously curved, unbranched, µm. Conidiogenous cells terminal, unbranched, medium brown, smooth, tapering to flat-tipped apical loci, proliferating sympodially, rarely percurrently near apex, µm. Conidia solitary, medium brown, smooth, prominently guttulate, obclavate, apex subobtuse, base short obconically truncate, straight to slightly curved, (0 ) 5 9( 12)-septate, (50 )80 120( 140) (5 )6( 7) µm; hila inconspicuous, 2 3 µm wide. Cultural characteristics: Colonies on MEA erumpent, raised, convex, with moderate aerial mycelium and feathery, uneven margins; on MEA surface pale olivaceous-grey, with patches of smoke-grey; outer margin olivaceousgrey to iron-grey; reverse iron-grey, reaching 20 mm diam after 2 months at 25 C. Specimen examined: Italy, Viterbo, Norchia, on leaves of Eucalyptus sp., Apr. 2005, W. Gams, holotype CBS-H 19841, cultures ex-type CPC = CBS , CPC Notes: The ITS sequence of P. norchiensis is identical to that of P. nogalesii, which was described from Chamaecytisus in New Zealand (Braun et al., 2003). It can be distinguished morphologically, however, by having 173

32 extremely dense caespituli, lacking superficial mycelium, having conidia that are more obclavate in shape, and also being larger and wider than those of P. nogalessii ( µm; Braun et al., 2003). Based on the key of Braun and Dick (2002), as well as recently described species (Crous et al., 2004b; Hunter et al., 2006a), P. norchiensis is morphologically distinct from the taxa presently known from Eucalyptus. Ramularia eucalypti Crous, sp. nov. (Fig. 17) MycoBank Etymology: Named after its host plant genus, Eucalyptus. Conidia catenulata, levia, hyalina, subcylindrica vel fusoideo-ellipsoidea, 0 1-septata, (10 )12 15( 18) (2.5 )3( 4) µm. Leaf spots amphigenous, irregular, subcircular or angular, confined by leaf veins, medium brown with a thin, red-brown border, specks 1 2 mm diam, or larger spots and blotches up to 4 cm diam. Mycelium internal and external, hyaline, smooth, consisting of branched, septate, hyphae, 3 4 µm wide. Caespituli fasciculate, amphigenous, hyaline, up to 80 µm wide and 50 µm high, situated on a poorly developed substomatal stroma, up to 40 µm wide. Conidiophores arising in dense fascicles from a subhyaline stroma (rarely separate on superficial mycelium), smooth, hyaline, 1 7-septate, subcylindrical, straight to geniculate-sinuous, unbranched or branched below, µm. Conidiogenous cells terminal or lateral, integrated, hyaline, smooth, tapering to flat-tipped apical loci, µm; scars darkened, refractive, thickened, µm wide. Conidia catenulate in branched chains, smooth, hyaline; ramiconidia subcylindrical to fusoid-ellipsoidal, 0 1-septate, (10 )12 15( 18) (2.5 )3( 4) µm; secondary conidia fusoid-ellipsoidal, occurring in branched chains of up to 15 µm long, (5 )6 7( 8) 3( 3.5) µm; hila darkened, thickened, refractive, up to 1 µm wide. Cultural characteristics: Colonies on MEA spreading, erumpent, convex with uneven, convoluted surface, radially striated, with sparse to moderate aerial mycelium and submerged, uneven, feathery margins; surface dirty white, reverse brown-vinaceous in centre, becoming fawn in middle zone, and brownvinaceous in outer region; reaching 20 mm after 2 months at 25 C. Specimens examined: Italy, Norchia, on living leaves of Eucalyptus grandiflora, Apr. 2006, W. Gams, holotype CBS-H 19832, culture ex-type CPC = CBS , CPC ; Viterbo, on living leaves of E. grandiflora, Apr. 2006, W. Gams, CPC = CBS , CPC Australia, Queensland, Cairns, Kuranda, Karoomba River Walk, S , E , on leaves of Eucalyptus sp., 19 Aug. 2006, P.W. Crous & J. Stone, CPC = CBS Notes: Presently this is the only true member of Ramularia known from Eucalyptus, as Ramularia pitereka and aggregate species are now accommodated in the genus Quambalaria (Quambalariaceae) (De Beer et al., 174

33 Fig. 17. Ramularia eucalypti (CBS H-19832). A, B. Leaf spots. C. Conidiophores. D F. Conidia in chains. Scale bars: C = 10, D = 6 µm. 2006). Ramularia eucalypti was collected from several locations in Italy, where it was associated with severe leaf spotting symptoms of mature Eucalyptus trees. It is interesting that the disease has not previously been reported from Australia, where eucalypts are native. Based on the species of Ramularia known from culture, R. eucalypti appears to be new, though further collections from other hosts will have to address the potential host specificity of this species. Currently Ramularia is accepted as being a host-specific genus of phytopathogenic fungi (Braun, 1998), though some exceptions are likely to emerge. Septoria sp. (Figs 18, 19) Leaf spots absent, conidiomata associated with leaf litter. Mycelium internal, consisting of smooth, branched, septate, pale brown, µm wide hyphae. Conidiomata pycnidial, immersed, brown, globose in OA, up to µm diam; wall consisting of 3 4 cell layers of textura angularis. Conidiogenous cells lining the inner layer of the conidioma, densely aggregated, ampulliform to subcylindrical, straight to curved, unbranched, 175

34 Fig. 18. Septoria sp. (CBS H-19831). A. Colony on OA. B, C. Conidia. Scale bar = 10 µm. Fig. 19. Conidia and conidiogenous cells of Septoria sp. (CBS H-19831). Scale bar = 10 µm. 176

35 hyaline, smooth, proliferating sympodially near the apex, µm. Conidia solitary, hyaline, smooth, finely guttulate or not, subcylindrical to narrowly obclavate, with subobtuse apex, and long subtruncate base, straight to curved, 1( 3)-septate, (9 )17 20( 24) 1.5( 2) µm; hila inconspicuous, µm diam. Cultural characteristics: Colonies erumpent, spreading, with even, lobate margins; on OA with moderate, dirty pink to white aerial mycelium, umber in outer region, which lacks aerial mycelium; reaching 35 mm diam after 1 month at 25 C; on PDA erumpent, central part with dense tufts of dirty white aerial mycelium, outer zone chestnut; reverse chestnut; reaching 25 mm diam after 1 month at 25 C. Specimen examined: Italy, Viterbo, Norchia, on leaves of Eucalyptus sp., Apr 2005, W. Gams, CBS-H 19831, cultures CPC = CBS , CPC Notes: Based on their ITS DNA sequence data, these isolates are similar to those of S. protearum Viljoen & Crous, known from Protea leaf spots in South Africa (Crous et al., 2004a). However, the conidia are somewhat narrower than those of S. protearum. Additional genes will therefore have to sequenced to fully resolve the status of the Eucalyptus isolates. Stenella eucalypti Crous & Summerell, sp. nov. (Fig. 20) MycoBank Etymology: Named after its host genus, Eucalyptus. Stenellae pseudoparkii similis, sed conidiis et conidiophoris longioribus distinguenda. Leaf spots amphigenous, irregular to angular specks, 1 3 mm diam, pale brown with dark brown, with raised, dark brown spots inside lesions, presumably due to insect damage; borders raised, margins absent to red-purple, but the latter may be due to co-colonization of a Pseudocercospora sp. Mycelium internal and external, consisting of branched, septate, medium brown, finely verruculose hyphae, 3 4 µm wide; terminal hyphal ends characteristically ending in clusters of globose, multi-celled chlamydospore-like structures, frequently surrounded by a mucus sheath; clusters µm diam. Conidiophores arising singly from superficial mycelium, dark brown, finely verruculose, multi-septate, subcylindrical, straight to geniculate-sinuous, mostly unbranched, or branched below, µm. Conidiogenous cells terminal, mostly unbranched, medium brown, smooth to finely verruculose, tapering to flat-tipped apical loci, proliferating sympodially, µm; scars thickened, darkened, refractive. Conidia solitary, pale brown, finely verruculose, guttulate, subcylindrical to narrowly obclavate, apex subobtuse, base long obconically subtruncate to obconically subtruncate, straight to slightly curved, (0 )1 3( 5)-septate, (10 )20 35( 60) (2 )3 4( 6) µm; hila thickened, darkened, refractive, µm wide. 177

36 Fig. 20. Stenella eucalypti (CBS H-19830). A. Leaf spot. B. Colony on MEA. C E. Conidiophores. F, G. Chlamydospore-like structures. H. Conidia. Scale bar = 10 µm. Cultural characteristics: Colonies on MEA reaching 15 mm diam after 2 months at 25 C; erumpent, with moderate aerial mycelium and smooth, uneven margins; surface mouse-grey to olivaceous-grey; reverse greenish-black. Specimen examined: Australia, Queensland, Cairns, Eureka Creek, 48 km from Mareeba, S , E , 468 m, on leaves of Eucalyptus tereticornis, 26 Aug. 2006, P.W. Crous, holotype CBS-H 19830, CPC = CBS Notes: Several species of Stenella are known from Eucalyptus (Crous, 1998; Crous et al., 2006g). Stenella eucalypti has conidia that are µm, 0 5-septate, showing some overlap with those of S. pseudoparkii ( µm, 1 5-septate) and S. xenoparkii ( µm, 1 2-septate), but is distinct in having somewhat longer and wider conidia, and very long conidiophores. The phylogenetic analysis could not confidently place this species; the parsimony analysis places it basal, whereas with neighbour-joining 178

37 it clustered with Phaeophleospora stonei. A Blast search with the ITS sequence reveals the highest similarity with species of Cercospora and Septoria. New and interesting records Mycosphaerella acaciigena Crous & M.J. Wingfield, Stud. Mycol. 50: Specimens examined: Australia, New South Wales, on leaves of Eucalyptus sp. Aug. 2006, B. Summerell, CPC = CBS , CPC Venezuela, El Piñal Lotes farm near Acarigua, on leaves of E. camaldulensis E. urophylla, Aug. 2006, M.J. Wingfield, CPC Notes: Mycosphaerella acaciigena was recently described from leaf spots on Acacia mangium leaves collected in Venezuela (Crous et al., 2004c). Although this is the first report of this fungus from Eucalyptus, and also the first report from Australia, several species of Mycosphaerella are now known to move between Eucalyptus and Acacia hosts (Crous and Groenewald, 2005). Mycosphaerella heimii Crous, S. African For. J. 172: Specimens examined: Australia, Queensland, Cairns, close to Kuranda, Kennedy Highway, S , E , on leaves of Eucalyptus platyphylla, 26 Aug. 2006, P.W. Crous, CPC = CBS , CPC Thailand, Thatakiab District, Chachoengsao Province, on leaves of E. camaldulensis, 12 Oct. 2006, W. Himaman, CPC = CBS , CPC Venezuela, on leaves of E. urophylla, Aug. 2006, M.J. Wingfield, CPC , , = CBS , CPC Notes: Since M. heimii was originally described from Eucalyptus leaves collected in Madagascar, it has been reported on this host from several countries (Crous, 1998), including a recent report from Australia (Whyte et al., 2005), which is confirmed by the present collection. Mycosphaerella konae Crous, Joanne E. Taylor & M.E. Palm, Mycotaxon 78: Anamorph: Pseudocercospora sp. Specimen examined: Thailand, Thatakiab District, Chachoengsao Province, on leaves of E. camaldulensis, 12 Oct. 2006, W. Himaman, CPC = CBS , CPC Notes: Mycosphaerella konae is known to be a pathogen of Banksia and Leucospermum spp. cultivated in Hawaii (Crous et al., 2004a). This is the first report of this fungus on Eucalyptus in Thailand. The present collection closely matches the type with regards to ascospore dimensions and germination patterns, and similar cultural characteristics. 179

38 Mycosphaerella mexicana Crous, Mycol. Mem. 21: Specimen examined: Hawaii, Waimea, ascomata occurring on older lesions of Aulographina eucalypti, on Eucalyptus leaves, Aug. 2005, W. Gams, CPC = CBS , CPC Notes: Mycosphaerella mexicana was originally described from eucalypt leaves collected in Mexico (Crous, 1998), and has subsequently been recorded from Australia (Maxwell et al., 2003). This is, however, the first report of this fungus from Hawaii. Although there are a few base pair differences compared to the sequences derived from the Australian cultures, sparse material made it difficult to compare morphologically to M. mexicana. Mycosphaerella molleriana (Thüm.) Lindau in Engler & Prantl., Natürlichen Pfanzenf. 1: (Fig. 21) (Basionym) Sphaerella molleriana Thüm., Revista Inst. Sci. Lit. Coimbra 28: Anamorph: Colletogloeopsis molleriana Crous & M.J. Wingf, Can. J. Bot. 75: Specimens examined: Portugal, Lusitania, leaves of E. globulus, Jul. 1879, Fr. Moller, K (holotype of teleomorph); Abrantes, leaves of E. globulus, Jul. 1995, S. McCrae, PREM (holotype of anamorph), cultures ex type CPC 1214 = CBS , CPC 1215 = CBS ); Lisbon, N , W , 77 m, on leaves of Eucalyptus sp., 13 Oct. 2006, P.W. Crous & A.J.L. Phillips, CBS-H 19826, epitype of teleomorph designated here, cultures ex-epitype CPC = CBS , CPC Notes: Crous and Wingfield (1997) described the anamorph of M. molleriana, and this culture has since been used as representative of the species. The present collection contains numerous ascomata, and is morphologically and genetically similar to the anamorph strain, while the morphology matches that observed on the holotype of the teleomorph. This fresh collection, which has ample fruiting of both states, can thus be used to epitypify the holomorph. Mycosphaerella ohnowa Crous & M.J. Wingf., Stud. Mycol. 50: Specimen examined. Australia, New South Wales, Dilkoon, Hourne Plantation, 153º 1 47 E, 29º S, on leaves of Eucalyptus dunnii, 12 Feb. 2006, A.J. Carnegie, CPC = CBS , CPC Notes: Mycosphaerella ohnowa is presently known to occur on E. grandis leaves in South Africa (Crous et al., 2004b), and this is the first record from Australia. The present collection agrees well with that of the type strain in cultural characteristics (colour, growth rate and slimy aerial hyphal tufts) and morphology. Mycosphaerella thailandica Crous, Himaman & M.J. Wingf., Stud. Mycol. 50: Anamorph: Pseudocercospora thailandica Crous, Himaman & M.J. Wingf., Stud. Mycol. 50:

39 Fig. 21. Mycosphaerella molleriana and its anamorph Colletogloeopsis molleriana (CBS H ). A. Leaf spot (arrows indicated subepidermal ascomata). B. Sporulation on MEA. C, D. Conidia and conidiogenous cells. E, F. Asci. G. Ascospores. H. Germinating ascospores. Scale bar = 10 µm. Specimen examined: Thailand, Thatakiab District, Chachoengsao Province, on leaves of E. camaldulensis, 12 Oct. 2006, W. Himaman, CPC 13477, , = CBS Notes: Mycosphaerella thailandica is associated with leaf spots of Acacia mangium in Thailand (Crous et al., 2004c). This is the first report of this fungus on Eucalyptus in Thailand. The present collection closely matches the type with regards to ascospore dimensions, germination patterns, and cultural characteristics. Cultures remained sterile, and did not produce the anamorph. Stigmina eucalypti Alcorn, Trans. Brit. Mycol. Soc. 60: (Fig. 22) Specimens examined: Australia, Queensland, Brisbane, on leaves of Eucalyptus tessellaris, 19 Nov. 1969, A. Skoien, holotype IMI ; Australia, New South Wales, Jackadgery, Singh Plantation (adjacent Inglebar State Forest), 152º E, 29º S, on leaves of Corymbia variegata, 11 Mar. 2006, G. Price, CBS-H 19834, cultures CPC = CBS 121, CPC

40 Fig. 22. Stigmina eucalypti (CBS H-19834). A, B. Colonies on MEA. C F. Conidiogenous cells. G, H. Conidia. Scale bars = 10 µm. Notes: A recent study by Crous et al. (2006c) confirmed Stigmina to be synonymous with Pseudocercospora and Phaeoisariopsis. Stigmina eucalypti, however, clusters apart from Stigmina s.str. (typified by S. platani). Because the generic affinity of S. eucalypti is uncertain, this species is tentatively retained in Stigmina until more molecular data become available. Acknowledgements We thank Drs W. Gams, A.J.L. Phillips, S. Mohali, I. Smith and M.J. Wingfield (MJW), who provided specimens without which this study would not have been possible. MJW is also thanked for comments on a draft of the script. Several technicians assisted with this project, namely A. van Iperen (cultures), M. Vermaas (photo plates), and M. Starink (DNA sequencing). Dr R.C. Summerbell is thanked for his comments on the morphology of the fungi named in the genus Cibiessia. 182